Changing Disease Course of Crimean-Congo Hemorrhagic Fever in Children, Turkey

Crimean-Congo hemorrhagic fever (CCHF), endemic in certain regions of the world, is listed as a priority disease with pandemic potential. Since CCHF was first identified in Turkey, children have been known to experience milder disease than adults. However, during the COVID-19 pandemic, we observed an unusually severe disease course, including hemophagocytic lymphohistiocytosis (HLH). We examined cytokine/chemokine profiles of 9/12 case-patients compared with healthy controls at 3 time intervals. Interferon pathway–related cytokines/chemokines, including interleukin (IL) 18, macrophage inflammatory protein 3α, and IL-33, were elevated, but tumor necrosis factor-α, IL-6, CXCL8 (formerly IL-8), and cytokines acting through C-C chemokine receptor 2 and CCR5 were lower among case-patients than controls. Interferon pathway activation and cytokines/chemokines acting through CCR2 and CCR5 improved health results among children with severe CCHF. Children can experience severe CCHF, including HLH, and HLH secondary to CCHF can be successfully treated with intravenous immunoglobulin and steroid therapy.

Crimean-Congo hemorrhagic fever (CCHF), endemic in certain regions of the world, is listed as a priority disease with pandemic potential. Since CCHF was first identified in Turkey, children have been known to experience milder disease than adults. However, during the COVID-19 pandemic, we observed an unusually severe disease course, including hemophagocytic lymphohistiocytosis (HLH). We examined cytokine/chemokine profiles of 9/12 case-patients compared with healthy controls at 3 time intervals. Interferon pathway-related cytokines/ chemokines, including interleukin (IL) 18, macrophage inflammatory protein 3α, and IL-33, were elevated, but tumor necrosis factor-α, IL-6, CXCL8 (formerly IL-8), and cytokines acting through C-C chemokine receptor 2 and CCR5 were lower among case-patients than controls. Interferon pathway activation and cytokines/chemokines acting through CCR2 and CCR5 improved health results among children with severe CCHF. Children can experience severe CCHF, including HLH, and HLH secondary to CCHF can be successfully treated with intravenous immunoglobulin and steroid therapy.
Although some pediatric patients have died, their cytokine profiles did not differ from those for adults or other children on the basis of disease severity (15).
Since 2015, CCHF has been considered one of the emerging infectious diseases most likely to cause major epidemics; at present, it is listed by the World Health Organization as a priority disease with pandemic potential (16). During the COVID-19 pandemic, we observed an increased number of pediatric CCHF cases in Turkey associated with unexpectedly severe disease, including certain cases referred to or misdiagnosed as multisystem inflammatory syndrome in children (MIS-C) (17). In this study, we aimed to explain the reasons for variations in disease severity among children by determining cytokine/chemokine profiles over time, as well as evaluating clinical and laboratory parameters of patients.

Study Design and Patients
We conducted a retrospective study of 12 patients with PCR-confirmed CCHF and 11 healthy volunteers as a control population during April 22, 2020-August 31, 2021, at Hacettepe University İhsan Doğramacı Children's Hospital in Ankara, Turkey. We obtained demographic information, self-reported times of tick bites and onset of symptoms, and results from physical examinations and diagnostic tests from patient medical records. We performed quantitative reverse transcription PCR (qRT-PCR) targeting CCHF virus for all patients at the Turkey Public Health Reference Laboratory (PHRL).
All 12 patients seeking treatment were diagnosed with HLH on the basis of criteria from the Histiocyte Society HLH-2004 study (18). We tested all CCHF patients for SARS-CoV-2 at admission by using qRT-PCR targeting SARS-CoV-2 with primers supplied by PHRL. The volunteer controls had no history, signs or symptoms, or physical findings of disease and no underlying conditions. Because the study was performed during the pandemic, we also tested controls for SARS-CoV-2 to eliminate the possibility of asymptomatic COVID-19. We obtained informed written consent from guardians of all patients and controls. Our study was approved by the Hacettepe University Ethics Committee (GO21/413).

Serum Collection and Laboratory Tests
For each CCHF patient, we measured complete blood count with differentials, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK), lactate dehydrogenase (LDH), ferritin, prothrombin time, activated partial thromboplastin time (aPTT), international normalized ratio (INR), fibrinogen, D-dimer, triglycerides, sodium, potassium, blood urea nitrogen, albumin, and glucose at admission and every 8-12 h until laboratory findings were at safe levels, then daily. At 8-12, 48-60, and 96-120 h after admission, we collected an additional 3 sets of serum samples in addition to the HLH marker samples, centrifuged them at 4,000 rpm for 10 min at room temperature, and stored them at −80°C until used for cytokine/chemokine profiling. We obtained blood samples from controls once and stored them at −80°C until used as group measures for cytokine/ chemokine profiling.

Statistical Analysis
We used IBM SPSS Statistics 26 (https://www.ibm. com) for all statistical analyses. We expressed categorical variables as frequencies and percentages and continuous variables as medians with interquartile ranges (IQRs). To compare cytokine/chemokine levels between patient and control groups, we used the Mann-Whitney U test and set p <0.05 as statistically significant. We performed Friedman 2-way analysis of variance of ranks to determine the changes in cytokine/ chemokine and laboratory parameter levels over the 3 time intervals. We calculated the time interval contributing to change using Pearson pairwise comparison with Bonferroni correction. Finally, we determined correlation between laboratory tests and cytokine/ chemokine levels with Pearson correlation analysis. We used GraphPad Prism version 9.2.0 (https://www. graphpad.com) for figure configurations.

Laboratory Findings
All patients had leukopenia, lymphopenia, neutropenia, and thrombocytopenia when initially seeking treatment; 10/12 (83%) had normal hemoglobin levels. Other laboratory findings varied at admission and each time interval (Table 2). We observed that all patients deteriorated clinically over the course of the first time interval, consistent with their worsening laboratory tests. Except for triglycerides, fibrinogen, ESR, ALT, and AST, all laboratory parameters were at their worst levels during the first time interval; ALT and AST levels were at their highest during the second time interval, and ESR, fibrinogen, and triglycerides reached their maximum levels during the third interval. Increases were not statistically significant for ALT (p = 0.76), AST (p = 0.138), or fibrinogen (p = 0.07) but were for both ESR (p = 0.016) and triglycerides (p = 0.001). Over the third time interval, neutrophil (p = 0.134) and lymphocyte (p = 0.105) counts did not differ significantly, but both platelet (p = 0.045) and erythrocytes (p = 0.05) counts increased significantly. All patients had a significant decline in hemoglobin levels during the third time interval (p = 0.001). By the third time interval, INR was within normal range for all patients and aPTT levels for 91.7% (11/12), but only 8.3% (1/12) of patients had normal D-dimer levels. Declines in all 3 coagulation parameters over time were significant (p = 0.005 for INR, p = 0.002 for aPTT, and p = 0.001 for D-dimer). Median CRP levels were within normal limits for all 3 intervals, but change over time was significant (p = 0.001). Serum ferritin, CK, and LDH levels peaked during the first time interval and declined significantly over time (p = 0.035 for ferritin, p = 0.01 for CK, and p = 0.005 for LDH). Although nearly half (5/12) of patients had normal CK values at the third time interval, LDH and ferritin levels were above normal in all patients. We performed bone marrow aspiration on 10/12 (83%) patients, all of whom exhibited hemophagocytosis within 24-48 hours after admission.

Treatment
We administered 2 g/kg body weight intravenous immunoglobulin to all patients within 12 hours of admission, either as a 48-hour infusion or 400-500 mg/kg/day over 4-5 days. As part of HLH treatment for all patients, we administered dexamethasone to 8 patients and methylprednisolone to 4. Initial doses were 10 mg/m 2 body surface area for dexamethasone and 2 mg/kg/day for methylprednisolone. Median ±SD initiation time for corticosteroid treatment was 25 ±3.86 hours (range 24-36 hours) and median duration of treatment was 8 ±2.57 days (range 3-10 days). We administered antiviral treatment (ribavirin) to all patients for a median of 5 ±2.54 days (range 2-10

Serum Cytokine/Chemokine Levels
Cytokine/chemokine values varied both over time and between patient and control groups (Table 3). CCL5 levels were not evaluated further after they measured above upper limits indicated by the manufacturer in 6/11 control patients.

Serum Cytokine/Chemokine Correlation with Laboratory Parameters
Some cytokines/chemokines were correlated with CCHF patient laboratory parameters depending on time interval (

Discussion
Since CCHF was first identified in Turkey, children have been observed to have milder disease (19,20). During the past 2 decades, studies have shown that the course and outcome of CCHF depend on viral load, host genetic factors, and host immune response, together with the level of release of cytokines/chemokines (10,12,14,21). Furthermore, the association of hemorrhagic fevers with HLH suggests the involvement of cytokines/chemokines in the pathogenesis of the disease (22)(23)(24)(25). One study reported HLH in 50% of adult CCHF patients (26); however, a case report describing an adult CCHF patient with HLH reported HLH as a rare condition (27). HLH is rarely reported among pediatric patients (28).
After the official announcement of the COV-ID-19 pandemic in Turkey on March 11, 2020, we began to observe certain changes in pediatric CCHF cases. The first noticeable change was admission of patients earlier in the year. Although ticks are known to emerge as early as March, the CCHF season in Turkey runs May-September, with June and July as the peak months (26,(29)(30)(31). During the study period, the first 2 cases were admitted in April, earlier than expected, probably because the patients had relocated to endemic rural valley districts as a result of restrictions and early school closures because of COVID-19.
A second change was observed in clinical manifestations. All 12 CCHF case-patients in the study manifested >1 severe disease sign fulfilling the criteria for HLH: all had fever and hepatosplenomegaly, headache was the next most common  complaint, most patients had petechiae, and 3 had neurologic findings at admission; none had tonsillopharyngitis. In previously published research on pediatric CCHF, the most common symptoms reported were fever and nausea, and tonsillopharyngitis was observed in a substantial percentage of patients, but hepatomegaly and splenomegaly were rarely reported (20,32).
Cytokines/chemokines play substantial roles in the pathogeny of viral hemorrhagic fevers, and previous work has proposed that CCHF cytokine profiles are similar to cytokine profiles of other hemorrhagic fevers (13,33). Studies on the cytokine and chemokine profiles of CCHF patients have mainly been conducted in adults, and the levels of the inflammatory cytokines IL-6 and CXCL8 have been proposed as correlated with severe and fatal CCHF (13). Contrary to those findings, in our study, neither IL-6 nor CXCL8 was elevated in any patient during any time interval. Our finding that IL-6 and TNF-α levels were not elevated was consistent with a previous pediatric study (15). However, the finding on IL-6 levels differed from another pediatric study in which pediatric CCHF patients were reported to have elevated levels of IL-6 and IL-10 compared with control groups (34). The difference might have been because of timing and a single measurement of cytokines/chemokines. In our study, IFN-α, IFN-γ, CXCL10, and CXCL11 were elevated during all 3 time intervals, indicating the consequential role of the T-helper 1-dependent pathway in pediatric CCHF cases. Certain strains of CCHF virus have been shown in vitro to delay IFN production (35) by suppressing IFN-β-promoter-mediated gene expression, hampering the interferon type 1 response (36). However, that pattern seems not to have been the case with the pediatric CCHF patients in our study, among whom the IFN-γ pathway was consistently active. IFN-γ induces production of CXCL10 and CXCL11, structurally associated chemokines that attract C-X-C chemokine receptor type 3 + (CXCR3 + ) lymphocytes expressed by T-helper 1 cells (37). Our findings suggest that CXCL10 and CXCL11 also modulate T-helper 1-adaptive immunity for the progression of CCHF. IL-18, a proinflammatory cytokine that was elevated during all 3 time intervals, induced IFN-γ production by CD4 and CD8 T-cells and natural killer cells. IL-33, another inducer of IFN-γ production, was elevated in the second and third time intervals. IL-17, which acts in concert with TNF-α and IL-1β and is inhibited by INF-γ, was not elevated in any time interval. The levels of CXCL5, a chemokine inhibited by INF-γ, were significantly low in all time intervals, further supporting INF-γ upregulation.
CCL2, which is expressed by macrophages in response to INF-γ, IL-6, TNF-α, IL-1β, and lipopolysaccharides, is another chemokine reported to correlate with the severity of CCHF (12,13). One study proposed that the course of disease in fatal cases was responsible for significantly high chemokines, mainly CCL2 in adult patients but not in pediatric patients, including patients with severe disease (38). This finding differs from our observation that all patients had high CCL2 levels during the first time interval; however, we did not observe elevated CCL2 levels in the subsequent intervals. MCP-1 binds to CCR2 and CCR5 receptors. Downregulation of CCR5 is known to be protective against HIV (39) and dengue virus (40); it has been reported that the lower the expression of CCR5, the better the outcome. Furthermore, proinflammatory cytokine CXCL1 acting through CCR2 levels was lower among patients than controls, suggesting that CCR2 receptors are downregulated in pediatric CCHF patients.
Correlations between cytokine/chemokine levels and laboratory parameters varied. Although all patients fulfilled the HLH criteria, triglyceride levels were invariably low during the first time interval. In that period, triglyceride levels negatively correlated with CXCL10 and CXCL11, both chemokines known to cause hypertriglyceridemia. Another notable laboratory parameter was CRP, which did not correlate with any cytokines/chemokines. A recent report described CCHF patients misdiagnosed with MIS-C during the pandemic (17). Indeed, 3 case-patients in our study were referred with a preliminary diagnosis of MIS-C. However, CRP levels differed uniquely between CCHF and MIS-C cases. CRP levels of almost all the CCHF cases were within reference limits throughout all time intervals, but median CRP levels in MIS-C cases were low, at 18.7 mg/dL in 1 study (41) and 19.6 mg/dL in another (42). Meanwhile, the CRP levels of the MIS-C cases associated with HLH were 7.5-21.9 mg/dL (43). Combined with a tick-bite history, low CRP levels should raise suspicion of CCHF in pediatric patients with HLH in endemic regions.
Among limitations of our study, the small number of CCHF patients, all of whom had severe disease, did not allow us to generalize findings to all CCHF patients. However, we believe that serial measurement of cytokines/chemokines in our study represents a notable advantage over studies that used a single measurement. Another limitation was that, although all patients had a cycle threshold value <20, it was not possible to have viral loads measured or CCHF virus strains sequenced because of increased laboratory workload during the COVID-19 pandemic.
In conclusion, our results show that children can have severe CCHF that manifests with HLHlike signs and symptoms. We observed strong IFN pathway activation and a T-helper 1-biased immune response. Downregulation of cytokines acting through CCR2 and CCR5 had a favorable effect among case-patients despite severe disease. In the future, larger controlled studies including CCHF pediatric patients with various disease severity should be conducted to verify the actual roles of different cytokine/chemokine profiles among children. Clinicians should be aware that children can manifest severe CCHF and that, in the context of the ongoing COVID-19 pandemic, those cases might be initially considered to be MIS-C. Our results further suggest that HLH secondary to CCHF can be successfully treated with intravenous immunoglobulin and steroid therapy.